Cholera is an often-fatal diarrheal disease caused by the bacterium Vibrio cholerae. This disease remains a health threat for the majority of the world, causing thousands of deaths every year. We have recently demonstrated that ToxT, the primary transcriptional activator of virulence genes in V. cholerae, is negatively regulated by certain environmental signals, and specifically by the presence of bile. Our studies will focus on dissecting the molecular mechanism(s) of environmental modulation of ToxT transcriptional activity, utilizing bile as an environmental modulatory factor. We wish to understand and exploit this negative regulation to develop novel means to prevent cholera. Essentially nothing is known about the structure/function of ToxT, so these studies also include the elucidation of the functions of the ToxT protein. Our approach first involves characterizing the domain structure of ToxT. This will be accomplished by (i). construction and characterization of chimeric ToxT proteins, and (ii). identification of ToxT amino acids important for DNA binding and transcriptional activation. Further characterization of ToxT will include the identification of all the ToxT-regulated genes of V. cholerae by microarray analysis, and the characterization of the ToxT DNA binding site(s). Once we have a more thorough understanding of ToxT, we will determine the mechanism of modulation of ToxT transcriptional activity by environmental signals, utilizing bile as the modulatory factor. These studies include: (i). determination of the effect of the porins OmpU and OmpT (which are known to be differentially permeable to bile) on bile modulation of ToxT activity, (ii). identification of additional V. cholerae genes involved in bile regulation of ToxT activity, (iii). Identification of ToxT amino acids necessary for bile regulation, and (iv). determination of the effects of bile on ToxT DNA binding activity. Finally, the relevance of environmental modulation of ToxT activity (by bile or other stimuli) will be assessed by testing the virulent properties of V. cholerae strains containing mutations that affect various aspects of ToxT transcription. Our ultimate goal is to learn how to manipulate ToxT by external factors in order to repress virulence gene expression and prevent cholera, i.e., to force V. cholerae to prevent itself from causing disease. This fundamentally different approach to cholera therapy could lead to novel antimicrobial strategies mimicking the effects of bile.